The morphogen concept is central to developmental biology. Morphogen molecules form concentration gradients to provide positional information to a field of cells. This application will focus on experimental studies to advance the morphogen concept. The Drosophila morphogen gradient of Bicoid (Bcd) instructs anterior- posterior (AP) patterning by directly activating its downstream target genes in a concentration-dependent manner. Although the role of Bcd in AP patterning is well established, the regulatory network controlling this process is not fully understood. For example, several aspects critical to the operation and regulation of the Bcd gradient system, e.g., Bcd degradation, post-translational modifications, and Bcd gradient scaling within a species, remain virtually unknown at this time. The overall hypothesis of this application is that a regulatory network enables Bcd to respond to various inputs, both before and during the time of gradient formation, and at the time of its action as a transcriptional activator. These three layers of the regulatory network are investigated in three specific aims. Aim 1 will study the mechanisms that regulate Bcd gradient formation by focusing on the role of MAP kinase (MAPK) and other regulators that control Bcd degradation. Aim 2 will investigate the mechanisms that regulate the action of Bcd as an activator by focusing on its interaction with MAPK and the roles of post-translational modifications. Aim 3 will investigate the mechanisms leading to a scaled Bcd gradient and will analyze the layer of regulation during oogenesis for its role in controlling the robust and scaled AP patterning later in embryos. The proposed study is based on strong preliminary studies and is made possible by a set of quantitative tools recently established in the lab. The proposed study will have powerful and sustained impact on our efforts to understand how morphogen gradients work and how developmental robustness is achieved. The proposed study is also important to human health because morphogens and MAPK signaling are fundamentally relevant to birth defects and, furthermore, the human homologue of a gene studied in this project has been implicated in otitis media, a leading cause of childhood deafness.